I. Histological Grading of Tumors
Prior to this invention, grading of some tumors, particularly of the breast, has been done by microscopic examination. In grading breast tumors, pathologists have traditionally used the Scarff-Bloom-Richardson system (see, Le Doussal, V., et al., Cancer 64(9):1914 (1989)). Although the S-B-R grading of tumors was an attempt at objective quantitation, microscopic tumor grading, by its nature, is subjective. Additionally, to grade tumors, the tumor or the cells from a tumor need to be removed. This requires surgical techniques.
Because of the subjective nature of tumor grading, the same pathologist should grade all the tumors. In addition, the pathologist should be well-trained in the grading of tumors by the S-B-R system. Alternatively, two pathologists can be used and the results obtained by each compared for consistency (Robbins, P., et al., Hum. Pathol. 26(8):873 (1995)).
Tumors can be graded histopathologically on many different bases. As mentioned above, for malignant breast tumors, grading systems such as S-B-R are preferred because they provide objective values of malignancy grade. The pathologist using the S-B-R system looks to three structural characteristics when grading tumors: (1) nuclear pleomorphism; (2) mitotic index; and (3) the ability of the tumor to form tubular, glandular or capillary formations, ie., ductoglandular differentiation (see, Le Doussal, supra). Tumors are graded by each criterion separately with I being the most normal (differentiated) and 3 the most aberrant (undifferentiated). The scores of the three criteria are added for a final tumor grade. Therefore, the scores can range from 3-5 (well differentiated) to 6-7 (moderately differentiated) and 8-9 (poorly differentiated).
II. Tumor Angiogenesis
Angiogenesis is the process by which new vessels grow toward and into a tissue. Angiogenesis is required for several physiologic processes including embryogenesis, corpus luteum formation, and wound healing. It is also a critical element in the pathogenesis of many disorders, most notably rapid growth and metastasis of solid tumors. With the current evolution of new strategies and drugs to intervene in the angiogenesis process, a complete understanding of angiogenesis has become clinically important.
The formation of new blood supplies is essential to the unrestricted growth of tumors. Tumors do not produce their own new blood vessels, but for nutrients and oxygen rely on vascular supplies derived from the nearby host tissue. It has been shown that tumors can attain a size of only 1-2 mm by simple diffusion of nutrients, but can exist for an extended period in this quiescent, static, prevascular stage before angiogenesis is "switched on" (Folkman, J., J. Nat'l Cancer Inst. 82:4 (1989)). Once angiogenesis is upregulated, the tumor enters the vascular phase allowing for exponential growth and resultant clinical manifestations. Although the mechanism of"switching on" is not known, once the switch has been thrown, angiogenic factors elaborated by the tumor and tumor-associated inflammatory cells interact with endothelial cells in neighboring capillaries to stimulate new capillary beds and to prepare the local environment for their ingrowth.
Being a limiting factor for both tumor growth and metastases, it has been assumed that angiogenesis correlates with tumor aggressiveness. This assumption has been supported in clinical trials investigating a variety of tumor types. Histologic assays of angiogenesis based on the microvascular density (MVD), ie., the number of endothelial clusters in a high-power microscopic field, in randomly selected human breast cancers showed that MVD correlated, as an independent factor, with the presence of metastases at time of diagnosis and with decreased patient survival times (Weidner, N., et al., N. Engl. J. Med. 324:1 (1991); Weidner, N., Am. J. Pathol. 147:9 (1995)).
However, histologic/pathologic grade is distinguished from angiogenic activity or from the more generic characteristic of tumor aggressiveness. Thus, functional characteristics, such as angiogenesis, and structural characteristics, such as markers of histologic grade, are considered independent tumor prognostic factors. See, Weidner, N., (1995), supra and Bevilacqua, P., et al., Breast Cancer Res. and Treatment 36:205 (1995) (intratumoral microvessel density is weakly associated with histological grade (P=0.053)).
II. Magnetic Resonance Imaging
The magnetic resonance imaging (MRI) scan is a diagnostic tool which is currently the most sensitive non-invasive way of imaging soft tissues of the body. Unlike a CT scan or conventional X-ray, this type of scanning device does not use radiation; instead, it makes use of magnetic fields which interact with the hydrogen atoms found in the water contained in all body tissues and fluids. Computers translate the increased energy of the hydrogen nuclei into cross-sectional images. The scanning procedure is very sensitive, and can often detect tumors that would be missed on a CT scan.
To increase the sensitivity of MRI as well as CT scans, contrast media are used. A contrast agent is typically a substance that is introduced into the body and accumulates preferentially in some tissues. In MRI, when a contrast agent comes in contact with the tissue, it changes the proton relaxation rate of the tissue. The magnetic resonance signals from those tissues can, therefore, be altered relative to other tissues with a lesser affinity to the contrast agent and become more visible by MRI.
Although "macromolecular MRI contrast media" (MMCM) have been known to those of skill for some time, see, Ogan, M.D., et al., Invest. Radiol. 22:665 (1987), these media only recently have found diagnostic uses. See, Kuwatsuru, R., et al., Magn. Reson. Med. 30:76 (1993). These media typically contain chelated gadolinium groups conjugated to proteins, such as albumin. These types of contrast agents, because they do not cross healthy blood vessel walls, have allowed investigators to gauge the endothelial permeability of tumor vessels compared to the permeability of vessels in healthy tissues.
III. Imaging of Tumors and Histopathological Grading
Herein, we describe a quantitative method to estimate the microvascular permeability of tumors, more particularly breast tumors, by macromolecular contrast media imaging (MCMI). We have previously shown that increased microvascular permeability correlates closely with increased microvascular density, a histologic surrogate of angiogenesis (van Dijke, C. F., et al., Radiology 198:813 (1996). Thus MCMI of tumors can be used to estimate the vascular permeability of tumors without the need for biopsy and angiogenic histologic evaluation.
Surprising results of this invention are the discoveries that microvascular permeability of tumors as measured by MCMI correlates with the determination of malignancy or non-malignancy and the S-B-R histological grading of malignant tumors. As stated above, microvascular density has not been thought to be a prognosticator of tumor grade and therefore malignancy. See, Weidner and Bevilacqua, supra. Thus, one of skill would not look to measuring angiogenesis or its surrogates by MCMI to predict the malignancy of tumors. However, as described below, tumor permeability as measured by MCMI correlated well (r.sup.2 .gtoreq.0.75) with S-B-R histology.
Because MCMI is a non-invasive technique and affords the possibility to grade inaccessible tumors, it is a powerful diagnostic in an oncologist's tool chest. In addition, this technique could find wide clinical application both for routine tumor evaluation (both for initial diagnosis and for follow-up) and for the conduct of clinical trials of new anticancer therapeutic agents.